Exposure of cells in tissue culture to inactivated herpes simplex virus 2 (HSV-2) results in a viral-induced altered phenotype. Mouse cells lacking thymidine kinase (tk) can integrate and express the viral tk gene, and rodent cells can become morphologically transformed. The proposed research investigates transformed cells with regard to which viral DNA sequences are retained, which genes are necessary for transformation, the arrangement of viral DNA, the synthesis of viral RNA, and the proteins for which the RNA codes. The studies will involve reassociation kinetics, Southern blotting, transformation with specific restriction endonuclease fragments, in situ hybridization, methyl-mercury gels and transfer to diazotized paper, cell-free translation of specific viral RNAs, and immunofluorescence and precipitation with specific antisera. The regulation of gene expression will be studied in both biochemical and morphological transformants. As a consequence of an initial HSV infection, certain cells, e.g., sensory ganglia, undergo an abortive infection in which the viral genome persists in a latent state. The state of the virus is presumed to be nonreplicating, but the genome may be replicating so slowly that detection of viral products has not been possible. This research will analyze viral transcription in latently-infected neurons by in situ hybridization with restriction endonuclease fragments as probes, to determine whether a specific RNA transcript is necessary for maintenance of the latent state.